Our overall goal is to understand the role of molecular chaperones in the clathrin-mediated endocytosis in mammalian cells, in the maintenance and propagation of yeast and mammalian prions, and in preventing aggregation of huntingtin fragments. To understand the role of molecular chaperones in clathrin-mediated endocytosis, we are using knockout mice models and the cells derived from the knockout mice. The mechanism of scrapie propagation is being studied by examining the trafficking of scrapie in chronically infected cell lines. The role of molecular chaperones in the propagation of yeast prions is being studied by combining light microscopy and plating assays. In addition, we have been using yeast as a model system to study the toxicity of huntingtin fragments. Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in these processes. We have continued our studies on the role of Hsc70 and the cochaperone GAK in clathrin-mediated endocytosis by determining which domains of GAK are required to restore normal clathrin-coated pit organization and clathrin-mediated endocytosis in GAK knockout mouse embryonic fibroblasts. These studies showed that a GAK fragment consisting of just the clathrin-binding domain and the J-domain by themselves completely rescues the defects caused by knocking out GAK. To determine whether these domains rescue the lethality observed by knocking out GAK in the mouse, a transgenic mouse was made that expressed the clathrin binding domain and J-domain of GAK. This truncated GAK fragment rescued lethality caused by knocking out GAK in either the brain or the liver of the mouse embryo. However, this transgene failed to rescue the lethality caused by knocking out GAK in the adult mouse. These results show that the kinase and pTen-like domains of GAK are essential for its cochaperone function in the adult mouse, but are not required for normal embryonic development. We also studied the propagation of prions, infective proteins that can misfold into an amyloid conformation both in mammalian and yeast cells. Prion propagation in mammalian cells was studied using chronically infected scrapie cells, which contain the misfolded amyloid form of the prion protein. To better understand the site of conversion of scrapie in the cell, different trafficking pathways for cargo were disrupted either by knocking down specific proteins or by expressing dominant negative Rab proteins. Scrapie localization was then determined by immunostaining, while the scrapie level was determined from Western blots. Surprisingly, blocking transport from the early endosome to the lysosome, the site of scrapie degradation, caused a marked reduction rather than an increase in scrapie levels. Apparently, the early endosome is a sorting station that determines whether scrapie either returns to the plasma membrane via the recycling endosome or to the lysosome via the late endosome. Evidently, altering traffic to the lysosome alters microdomains in the early endosome that both prevents conversion of the prion protein to scrapie and prevents the scrapie that is formed from trafficking to the recycling endosome and back to the plasma membrane If altering the microenvironment of the early endosome indeed prevents the conversion of the properly folded prion protein to the amyloid form this in itself could lead to the curing of scrapie. Our research on prion protein has also been extended to yeast, which provides a simple model system for studying prion propagation. In yeast, the molecular chaperone, Hsp104, regulates the inheritance of several yeast prions including PSI+, which is the prion form of the translation termination factor Sup35. Inactivation of Hsp104 cures all yeast prion proteins, but only PSI+ is cured by overexpression of Hsp104. Both the kinetics and the distribution of GFP-labeled Sup35 (NGMC) foci in the yeast population during PSI+ curing by overexpression of Hsp104 indicate that the mechanism of curing here is very different from the mechanism of curing by Hsp104 inactivation. Two models have been proposed to explain curing by overexpression of Hsp104. In one model overexpression of Hsp104 causes disaggregation of the prion seeds, while in the other model, there is asymmetric segregation of the prion seeds when the yeast divides. Using confocal imaging to follow the prion seeds during curing of PSI+, we found that the seeds were not retained in the mother as suggested by the asymmetric model. Sorting of mother and daughter cells by age also showed that the curing of PSI+ was independent of age again disproving the asymmetric segregation model. Rather, confocal imaging showed dissolution of the prion seeds during curing of PSI+ by Hsp104 overexpression suggesting that Hsp104 actually causes disaggregation of the prion seeds. Interestingly, the presence of misfolded prion protein causes the expression of huntingtin fragments with expanded polyglutamine repeats to be toxic in yeast. The toxic effect of the huntingtin fragments was affected by the PSI+ variant; the huntingtin fragments (Htt) were much more toxic in the strong variant than the weak variant. Since the strong PSI+ variant has much less soluble Sup35 than the weak variant, this suggested that toxicity might be caused by the Htt aggregate sequestering Sup35, which is an essential yeast protein. To determine whether sequestration of Sup35 was indeed causing toxicity in the cells with Htt aggregates, the yeast were transformed with a plasmid that expressed the soluble C-terminal fragment of Sup35, which by itself mediates translation termination. Expression of the C-terminal fragment of Sup35 almost completely rescued Htt toxicity, which shows that sequestration of Sup35 by huntingtin aggregates indeed caused toxicity. This is the first defined protein whose sequestration by Htt fragments produces toxicity in yeast.